The invention relates to a microplate, kit and testing method for a susceptibility testing of antibacterial drugs, especially antifungals.
With the increase of a crisis frequency of deep mycosis due to a yeast-like fungus and the diversification of casual bacteria, and the appearance of a bacterial strain showing the resistance to antifungal agents, an therapeutic drug appropriate for the infection disease is indispensable, and thus the necessity of a susceptibility testing of antifungal drugs has increased in medical services. As for the sensitivity testing method of yeast-like fungi, in 1992 Re Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Proposed Standards (M27-P), and in 1995 Tentative Standard (M27-T) were reported by the National Committee for Clinical Laboratory Standards (NCCLS) in United States, and Approved Standards (M27-A) was recommended in June, 1997. The M27-A method is a microdilution method in 0.2 ml of the medium amount, and is a method in which the bacterial growth inhibition concentration is read by a visual observation on the turbidity of a bacterial growth end point associated with the bacterial growth usually after the incubatation in the air for 46-50 hrs.
On the other hand, in Japan an independently improved microdilution method based on the above M27-P method was reported by the Standardization Committee in Japanese Society of Medical Mycology (Yamaguchi et al., Japanese Journal of Medical Mycology Society, 36; 61-86, 1995).
However, the NCCLS M27-A and Japanese Society for Medical Mmycology methods contain the following problems in case of an operation in clinical examination services.
1) It takes time for the preparation and the separate infusion. 2) It is difficult to read the bacterial growth end point (80% bacterial growth inhibition) in an azole type drug by the naked eye, and reading the bacterial growth end point requires to be fully accustomed. 3) In case of reading byamicroplate reader, it is difficult for packets to disperse uniformly, and it happens to read even a minute packet, consequently resulting to a reason for the decrease of reliability, etc.
Owing to these, in clinical examination services a testing method which can be carried out by a simpler operation than those reported currently and easily give a reliable reading result, and the reagent development for it are waited. It maybe considered that a reagent favorable for aperson carrying out the testing has a property in which the drug susceptibility testing can be started only by separately inoculating a bacterial suspension of a sample onto a microplate and the bacterial growth end point can easily be read after incubating this for a certain time. However, the bacterial growth end point that is read is required not to largely depart from that by the NCCLS M-27 method.
It can be considered that the development of a microplate in which a testing reagent is previously made into concentration dilution series contributes to the simplification of a drug susceptibility testing. However, owing to the fact that the bacterial growth end point is read from the degree of the turbidity in case of separately infusing a bacterial suspension of a sample onto said microplate, the above problem at the time of reading remains without any solution. As a reading method of the bacterial growth end point without depending on the turbidity, by using (Alamar Biosciences Inc., Sacramento, Calif.), a redox indicator, the method to read from the change of color-tone in the medium (Pfaller, M. A. et. al., J. Clin. Microbiol. 32: 506-509, 1994; Pfaller, M. A. et. al., J. Clin. Microbiol. 32: 1625-1628, 1994) and the method to read by using resazurin as an indicator (Yamane et. al., Rinsyo Byori (in Japanese), 44; 67-75, 1996) are reported. However, in these methods the procedure of adding the indicators to the culture liquid or the bacterial culture liquid is necessary. Further, these method become difficult for reading in the case that the medium shows an intermediate color, and additionally there is the handling problem that the medium mixed with resazurin must be stored and incubated under dark owing to the instability to light (JP, A, H9-127097).
The inventors applied the method of reducing the tetrazolium salt and measuring the amount of formazan (color) thus resulted (Formazan color method: Mosmann, T., J. Immunol. Methods, 65, 55-63, 1983) to the measurement of the growth rate of yeast-like fungi, and found that there was a correlation between the absorbancy and the turbidity (9th. Japanese Society for Clinical Microbiology Meeting: Jan. 31-Feb. 1, 1998). Then, the inventors tried to develop a simple determining agent, i.e. an agent with which the drug susceptibility testing could be started only by separately infusing a fungal suspension of a sample onto a microplate, and which could easily determine a fungal growth end point after incubating it for a certain period of time, by applying the above-mentioned method of measuring into a drug susceptibility testing. Specifically, the inventors produced a microplate wherein the drug and the color reagent were premixed in wells of the microplate and the mixed reagents were formed into a solid phase by a vacuum drying. However, in the step of drying, formazan was formed in wells of the microplate and the microplate could not be used for a drug susceptibility testing since the formazan caused experimental errors. Therefore, a microplate wherein both a drug and a color reagent were formed into a solid phase could not appropriately be produced.
However, as a result of further investigation to develop the above-mentioned simple determining agent, the inventors found that the formation of formazan in the step of drying is caused by a reaction of a color reagent with dimethyl sulfoxide (DMSO) in a drug solution, which is concentrated during the step of drying. Further, the inventors found that a microplate wherein both a drug and a color reagent can be formed into a solid phase without the formation of formazan by following steps: (1) separately infusing a drug solved with DMSO or a DMSO contained solution into wells of a microplate, (2) evaporating DMSO from the wells by drying under reduced pressure, (3) adding a color reagent into the wells, and (4) drying under reduced pressure again. Finally, the inventors confirmed that a drug susceptibility testing can easily be carried out by using the microplate produced without formation of formazan and the present invention has been completed. Further, the inventors found that the color reaction due to medium components can be suppressed by concurrently forming potassium ferricyanide and potassium ferrocyanide into a solid phase.
Namely, the invention is to provide a microplate for a drug susceptibility testing wherein a drug and a color reagent are formed into a solid phase on the microplate by twice vacuum drying steps. Preferably, the microplate is substantially free of formazan. According to a more preferred embodiment, the microplate is free of formazan.
Also, the invention is to provide a microplate for a drug susceptibility testing wherein the above color reagent contains a tetrazolium salt, 1-methoxy-5-methylphenazinium methylsulfate (hereafter described as 1-methoxy PMS), potassium ferricyanide and potassium ferrocyanide, and a kit product consisting of the microplate and a medium for a cell growth.
Further, the invention is to provide a drug susceptibility testing method comprising the following steps.
(1) Preparing a microplate wherein a drug and a reagent which contains a tetrazolium salt, 1-methoxy-5-methylphenazinium methylsulfate, potassium ferricyanide and potassium ferrocyanide are formed into a solid phase on the microplate by twice vacuum drying steps;
(2) adding a sample containing cells to the above microplate;
(3) incubating the above microplate; and
(4) reading the growth rate of the cells by the coloration rate.
In the following, the invention will be explained in detail.
The preparation of the microplate of the invention and the drug susceptibility testing method by its use were carried out by the method described in the following.
A. Preparation Method for Microplate with Drug and Color Reagent Formed into Solid Phase
For the solid phase formation on a microplate, a drug solution is dissolved in dimethyl sulfoxide (DMSO). By the use of the prepared drug a two fold dilution series is prepared in an appropriate concentration range with DMSO or a distilled sterile water, and a fixed amount is separately infused into each well and formed into the dry and solid phase under vacuum for 24 hrs. As the concentration range of a drug formed into the solid phase are cited a range such as, for example, Amphotericine B (AMPH): 16-0.03 xcexcg/ml, Flucytosine (5-FC): 64-0.125 xcexcg/ml, Fluconazole (FLCZ): 64-0.125 xcexcg/ml,Miconazole (MCZ): 32-0.06 xcexcg/ml and Itraconazole (ITCZ): 16-0.03 xcexcg/ml. After finishing the solid phase formation of a drug, the color reagent (containing a tetrazolium salt, 1-methoxy PMS, potassium ferricyanide and potassium ferrocyanide) is separately infused into each well and formed into a solid phase by a vacuum drying. The known tetrazolium salts showing a color by the reduction of the tetrazolium salts can be used, though appropriately is used a tetrazolium salt in which favorably the formed formazan pigment is easily soluble in water and the cell toxicity is low, for example, 4-[3-(2-methoxy-4-nitrophenyl)-2(4-nitrophenyl)-2H-5-tetr azolino]-1,3-benzene disulfonate sodium salt (WST-8; manufactured by Dojindo Laboratories, Ishiyama, M. et., al., Talanta, 44: 1299-1305, 1997). The prepared plate can be stored at 4xc2x0 C.
B. Antifungal Susceptibility Testing by using Plate with Solid Phase Formation of Drug
1) Medium
A medium for testing is prepared as follows. RPMI 1640 powder medium (with L-gulutamic acid, without NaHCO3, without phenol red) 10.4 g, glucose 10.0 g, NaHCO3 2.0 g and morpholinopropanesulfonic acid (MOPS) 34.53 g are added to a pure water 800 ml, and dissolved. The solution is adjusted to pH 7.0 by 1N NaOH. The total volume of the solution is adjusted to 1000 ml and the solution is sterilized by filtration. The medium thus prepared can be stored at 4xc2x0 C.
2) Fungal Strain
As a fungal strain for the application of the method are cited Candida genera such as Candida albicans, Candida parapsilosis, Candida tropicalis and Candida glabrata, and their relative yeast fungi, and especially a fungal strain which grows sufficiently in Sabourand-dextrose agar medium at 35xc2x0 C. for 24 hrs. is made the object.
As a fungal strain for an accuracy control are used Candida albicans ATCC 90028, Candida albicans ATCC 24433, Candida parapsilosis ATCC 90018, Candida parapsilosis ATCC 22019, Candida tropicalis ATCC 750, Candida krusei ATCC 6258, etc.
3) Testing method
The trace suspension dilution method of the invention is carried out as follows.
(1) A colony of a fresh isolate grown in an agar medium is used. Further, in case of using a stock strain the pre-incubation is done two times in an agar medium, followed by the usage. (2) A fungal suspension of the McFarland # 0.5 tubidity is prepared by the use of the fungal colony.
(2)-1. Three to five fungal colonies are inoculated in a test tube poured with a sterile saline 5 ml, and suspended. After suspending the suspension is thoroughly stirred by a vortex mixer so that it becomes homogeneous. The sterile saline is prepared by dissolving NaCl 8.5 g in the pure water 1000 ml and sterilizing in an autoclave (121xc2x0 C., 15 min.).
(2)-2. The fungal suspension is adjusted to the turbidity corresponding to McFarland # 0.5.
(3) The fungal suspension prepared in (2) is taken by a micropipete, added to the medium for the testing, thoroughly stirred by a vortex mixer and diluted so that the fixed concentration is obtained.
(4) The fungal suspension prepared in (3) is separately inoculated in a fixed: amount into a well in which a drug and a color reagent are formed into a solid phase. The replication of the fungal suspension is done within 15 min. after the fungal suspension preparation (in the case that the inoculation can not be done, it can be stored at 4xc2x0 C. within 2 hrs.). At the same time, as a negative control it is made that the medium for the testing is separately infused in a fixed amount into a well in which only a color reagent is formed into a solid phase, and as a positive control it is made that the fungal suspension prepared in (3) is separately infused in a fixed amount into a well in which only a color reagent is formed into a solid phase.
(5) After lidding the plate an aerobic culture is done at 35xc2x11xc2x0 C.
4) Reading method
The reading is carried out by the measurement values at a 24 or 48 hrs. incubation.
After stirring the microplate for 1 min., the absorbancy of the dominant wave length 450 nm and the complementary wave length 630 nm is measured, and the reading is carried out by the following method.
1. The minimum concentration showing the absorbency in the complete growth inhibition equal to or less than that in the negative control is made the minimum growth inhibition concentration (MIC).
2. The drug concentration of a well showing the absorbency equal to or less than the 80% growth inhibition concentration (IC 80) obtained by the following equation is made MIC.
IC80=(positive controlxe2x88x92negative control)xc3x970.2+negative control
C. Composition of Kit
In the kit of the invention are included a microplate prepared by the vacuum drying method as described above in which a drug and a color reagent are formed into a solid phase, and a medium for the testing. Namely, as an example of the kit of the invention is cited an antifungal susceptibility testing kit containing a microplate in which an appropriate antifungal such as AMPH, 5-FC, FLCZ, MCZ or ITCZ, and a color reagent are formed into a solid phase, and as a medium for the testing are cited glucose of 1.2%, MOPS buffered PRMI 1640 medium, and a sterile saline.